Flavonoids are major components of dietary supplements and herbal medicines with reported protective activities against microbial infections in humans. However, the precise mechanisms by which these plant derived natural products attenuate microbial infections are unclear. Using a high-throughput screen for type III protein secretion in Gram-negative bacterial pathogens, we discovered specific flavonoids from medicinal plants that do not interfere with bacterial growth can effectively antagonize this key bacterial virulence pathway and prevent Salmonella typhimurium invasion of host cells. To determine how specific flavonoids can antagonize type III secretion systems (T3SSs) and attenuate bacterial virulence, this application will 1) develop more potent flavonoid analogs, 2) determine flavonoid T3SS inhibitor mechanism of action and 3) analyze more active flavonoids in cellular and animals of S. typhimurium infection. As many Gram-negative bacterial pathogens use type III protein secretion systems to infection host cells, elucidating the mechanisms by which flavonoids inhibit bacterial virulence should reveal new targets and lead compounds that could be used to selectively target pathogenic bacteria and preserve beneficial host microbiota. Given the emergence of new and antibiotic-resistant bacterial pathogens, our studies should afford new anti-infective agents combat bacterial infections in humans. PUBLIC HEALTH RELEVANCE: The potential beneficial effects of flavonoids on human health has been extensively explored in the context of antioxidants, anti-inflammatory and anti-cancer agents and resulted in their distribution as dietary supplements. Our analysis of TCMEs that have been historically used as anti-infective remedies has revealed the unexpected and exciting discovery that specific flavonoids can antagonize T3SSs in bacterial pathogens and attenuate their virulence. The multi-disciplinary studies described in this application should help elucidate the flavonoid mechanisms of action on T3SSs and afford new lead compounds to attenuate bacterial pathogens in vivo. This approach should preserve the beneficial members of the host microbiota and minimize the development of antibiotic resistance to new anti-infective agents.